Changes in gene regulation may play an important role in both adaptive evolution and genetic disease. Current knowledge of cis-regulatory sequences is based largely on canonical transcription factor binding sites that affect gene expression levels or developmental patterns at specific time-points. Yet, temporal changes in gene expression may often be more important to fitness than steady-state levels. The goal of the proposed research is to understand which noncoding mutations alter gene expression and which changes in gene expression have phenotypic consequences. To accomplish this goal, cis-regulatory sequence function and evolution will be investigated in budding yeast by first identifying cis-regulatory changes that affect fitness and then measuring their effects on gene expression. Fitness assays will be used because they require no assumption as to which mutations alter cis-regulatory sequence function, or how they affect gene expression. Changes in cis-regulatory sequences that affect fitness will be related to canonical binding sites and to their effects on expression dynamics and steady-state levels. Dissection of these relationships will enhance our ability to identify cis- regulatory mutations that underlie phenotypic variation and will help define the role of expression dynamics in the evolution of gene regulation.